Spectroscopic Studies of Copper, Silver and Gold-Metallothioneins

Metallothionein is a ubiquitous protein with a wide range of proposed physiological roles, including the transport, storage and detoxification of essential and nonessential trace metals. The amino acid sequence of isoform 2a of rabbit liver metallothionein, the isoform used in our spectroscopic studies, includes 20 cysteinyl groups out of 62 amino acids. Metallothioneins in general represent an impressive chelating agent for a wide range of metals. Structural studies carried out by a number of research groups (using (1)H and (113)Cd NMR, X-ray crystallography, more recently EXAFS, as well as optical spectroscopy) have established that there are three structural motifs for metal binding to mammalian metallothioneins. These three structures are defined by metal to protein stoichiometric ratios, which we believe specifically determine the coordination geometry adopted by the metal in the metal binding site at that metal to protein molar ratio. Tetrahedral geometry is associated with the thiolate coordination of the metals in the M(7)-MT species, for M = Zn(II), Cd(II), and possibly also Hg(II), trigonal coordination is proposed in the M(11-12)-MT species, for M = Ag(I), Cu(I), and possibly also Hg(II), and digonal coordination is proposed for the metal in the M(17-18)-MT species for M = Hg(II), and Ag(I). The M(7)-MT species has been completely characterized for M = Cd(II) and Zn(II). (113)Cd NMR spectroscopic and x-ray crystallographic data show that mammalian Cd(7)-MT and Zn(7)-MT have a two domain structure, with metal-thiolate clusters of the form M(4)(S(cys))(11) (the alpha domain) and M(3)(S(cys))(9) (the beta domain). A similar two domain structure involving Cu(6)(S(cys))(11) (alpha) and Cu(6)(S(cys))(9) (beta) copper-thiolate clusters has been proposed for the Cu(12)-MT species. Copper-, silver- and gold-containing metallothioneins luminesce in the 500-600 nm region from excited triplet, metal-based states that are populated by absorption into the 260-300 nm region of the metal-thiolate charge transfer states. The luminescence spectrum provides a very sensitive probe of the metal-thiolate cluster structures that form when Ag(I), Au(I), and Cu(I) are added to metallothionein. CD spectroscopy has been used in our laboratory to probe the formation of species that exhibit well-defined three-dimensional structures. Saturation of the optical signals during titrations of MT with Cu(I) or Ag(I) clearly show formation of unique metal-thiolate structures at specific metal:protein ratios. However, we have proposed that these M=7, 12 and 18 structures form within a continuum of stoichiometries. Compounds prepared at these specific molar ratios have been examined by X-ray Absorption Spectroscopy (XAS) and bond lengths have been determined for the metal-thiolate clusters through the EXAFS technique. The stoichiometric ratio data from the optical experiments and the bond lengths from the XAS experiments are used to propose structures for the metal-thiolate binding site with reference to known inorganic metal-thiolate compounds.